Sperm competent for fertilization can become capacitated, bind to the zona pellucida (ZP) of an egg in a specific manner, and complete acrosomal exocytosis. Failure to carry out these functions results in infertility. Although the interactions between the ZP and the plasma membrane overlying the sperm acrosome have been considered important for sperm-egg recognition and signaling, recent results have prompted a reassessment of current paradigms concerning these interactions. The broad, long-term objective of this proposal is to understand how the acrosome functions in fertilization. The general hypothesis is that acrosomal exocytosis leads to the exposure of acrosomal matrix proteins that become a de facto extracellular matrix (ECM) on the surface of the sperm head, and that the dynamic interactions of this newly-exposed sperm ECM with the egg ECM (the ZP) govern sperm-egg recognition and sperm penetration of the ZP. Specific Aim 1 is to investigate acrosomal exocytosis using novel, sensitive analytical approaches to monitor OAM and PM fusion events in live sperm. The possibility will be examined that spontaneous acrosomal exocytosis is initiated during capacitation, a poorly defined maturation process that normally takes place within the female reproductive tract but can be mimicked experimentally. The hypothesis is that acrosomal exocytosis is initiated during capacitation and, in the absence of stimulatory factors, is a slow, discontinuous and Ca- dependent process. However, in the presence of stimulatory factors (e.g., ZP), acrosomal exocytosis is completed at an accelerated rate. Specific Aim 2 is to characterize the interactions of the acrosomal matrix with the zonae pellucidae of mammalian eggs. Components of the AM will be studied to determine if they possess ligand-binding properties that enable them to bind to the ZP of unfertilized eggs. Specific Aim 3 is to determine how structural properties and exocytosis-associated processing of AM components, e.g., sp56, affect their function. Structural determinants of sp56 will be studied to learn which domains are essential for binding of sperm to ZP glycoproteins by examining the structure-function relationships of mutated recombinant sp56 proteins. In addition, mutant mice null for the sp56 gene will be created to determine if sp56 is essential for fertilization. The proposed experiments will develop new technologies for the study of molecular mechanisms of acrosomal exocytosis. Results from these experiments may provide new ways to address the poor ZP binding of sperm from some human infertility patients and may offer new avenues for contraception through the disruption of purposeful sperm-ZP binding.